Abstract

Despite correlations between histone methyltransferase (HMT) activity and gene regulation, direct evidence that HMT activity is responsible for gene activation is sparse. We address the role of the HMT activity for MLL1, a histone H3 lysine 4 (H3K4) methyltransferase critical for maintaining hematopoietic stem cells (HSCs). Here, we show that the SET domain, and thus HMT activity of MLL1, is dispensable for maintaining HSCs and supporting leukemogenesis driven by the MLL-AF9 fusion oncoprotein. Upon Mll1 deletion, histone H4 lysine 16 (H4K16) acetylation is selectively depleted at MLL1 target genes in conjunction with reduced transcription. Surprisingly, inhibition of SIRT1 is sufficient to prevent the loss of H4K16 acetylation and the reduction in MLL1 target gene expression. Thus, recruited MOF activity, and not the intrinsic HMT activity of MLL1, is central for the maintenance of HSC target genes. In addition, this work reveals a role for SIRT1 in opposing MLL1 function.

Loss of MLL1 in Hematopoietic Cells Does Not Affect H3K4me Enrichment at Target Genes

(A) Transcripts corresponding to three representative MLL1 target genes decrease rapidly upon 4-OHT-induced excision of Mll1 in vitro. Linneg cells were purified from control ERT2-cre;Mll1F/+ or ERT2-cre;Mll1F/F bone marrow and were cultured in vitro in medium containing 400 nM4-OHT. Triplicate samples were harvested every 12 hr for real-time qPCR (A) and ChIP (C–E) assays.(B) Diagram of target gene loci showing ChIP amplicon location, approximately 1 kb to either side of the TSS (see ).(C–E) Anti-H3K4me1, -H3K4me2, and -H3K4me3 ChIP assays were performed using samples from the 48 hr time point. Enrichment of the indicated histone modification is shown for each amplicon as the mean of four PCRs ±SEM. ChIP results are representative of at least three independent experiments.

(A) Representative ChIP-seq tracks showing reads per million (y axis). LSK cells were sorted from control ERT2-cre;Mll1F/+ or ERT2-cre;Mll1F/F animals and cultured in vitro in HSC medium 48 hr (300 nM 4-OHT for only the first 24 hr). Complete deletion of Mll1 was confirmed (). The locus is diagrammed below the tracks.(B and C) Scatterplots showing the concordance between enriched regions in a representative pair of control versus Mll1Δ/Δ samples. Each dot represents enrichment of the indicated histone modification within a bin of 1 kb. Differentially enriched bins in control Mll1Δ/+ LSK cells are shown in green and those enriched in Mll1Δ/Δ LSK cells are shown in red. Pairwise comparisons between different replicate pairs yielded similar results and concordance between replicates is shown in .

ChIP assays performed 48 hr after initiating Mll1 deletion as in . Nuclei from linneg cells were fixed and ChIP performed using anti-H4K16Ac (A), -MOF (B), -Brd4 (C), -Cdk9 (D), -phospho-ser2 Pol II (E), and phospho-ser5 Pol II (F). Amplicons are as diagrammed in . Data represent mean of two to four replicates ±SEM and are representative of at least three experiments. ND, not determined.

ERT2-cre;Mll1F/+ or ERT2-cre;Mll1F/F cells were cultured as in to initiate Mll1 deletion. Duplicate ERT2-cre;Mll1F/F samples were incubated with both 4-OHT and either 25 mM Ex-527 (A) or 5 mM nicotinamide (B). After 48 or 60 hr, Mll1 target gene expression was determined by real-time qPCR. Means of four PCRs ±SEM are shown. Data are representative of two to three independent experiments.